Chlamydia trachomatis and other chlamydial species are important agents of ocular, genital tract, respiratory, and other infections in humans. The pathology of infection is thought to be associated with hypersensitive immune response in some diseases and, quite simply, the ability of chalamdiae to grow inside of and kill host cells at some stage of al diseases. The specifics virulence mechanisms that allow chlamydia to grow intracellularly,however, have not been identified. The long term goal of the laboratory continues to be, therefore, understanding how chlamydia grow inside of host cells. The objectives outlines in this proposal are to understand selected key events that take place early and late in the chlamydial cycle. Early in the chlamdial life cycle, the infectious form (EB) converts from a metabolically inter state to a form (RB) eventually capable of multiplication inside of a host cell. Using in vitro transcription and other molecular techniques developed during the past grant period, the cause of RB dormancy will be determined, and the genomic target and function of a DNA binding protein that is made within the first hour of infection and which may play a critical role in the regulation of the developmental cycle will be identified (Aim1). These studies should provide insight into how chlamydiae initiate infections and may ultimately lead to novel strategies for preventing chlamydial growth. Under Aims 2 and 3, the events that take place late in the life cycle, when RB's reorganize to EB's, will be examined. The regulation of a key late-stage operon encoding cysteine-rich proteins will be examined in Aim 2, and the mechanisms by which the envelope proteins will be examined in Aim 2, and the mechanisms by which the envelope proteins become cross-linked and the topological relationship among envelope proteins will be investigated under Aim 3. These studies should provide useful basic information about the envelope structure of chlamydia, which is unique among all bacteria and which allows EB's to survive extracellular environments and to attach to host cells. It is anticipated that new envelope proteins will identified and that new relationships and chlamdyial surface proteins will be revealed in our studies. Increased knowledge of the structure of the chlamydial cell envelope will facilitate our understanding of how these fascinating, unusual bacteria attach to host cells and may identify new surface-exposed protein complexes as potential subunit vaccine candidates.